NMR Relaxation Data Research Articles

Structure and Dynamics of the Homodimeric Dynein Light Chain km23

km23 (96 residues, 11 kDa) is the mammalian ortholog of Drosophila roadblock, the founding member of LC7/robl/km23 class of dynein light chains. km23 has been shown to be serine-phosphorylated following TGFbeta receptor activation and to bind the dynein intermediate chain in response to such phosphorylation. Here, we report the three-dimensional solution structure of km23, which is shown to be that of a homodimer, similar to that observed for the heterodimeric complex formed between p14 and MP1, two distantly related members of the MglB/robl superfamily, but distinct from the LC8 and Tctex-1 classes of dynein light chains, which also adopt homodimeric structures. The conserved surface residues of km23, including three serine residues, are located predominantly on a single face of the molecule. Adjacent to this face is a large cleft formed by the incomplete overlap of loops from opposite monomers. As shown by NMR relaxation data collected at two fields, several cleft residues are flexible on the ns-ps and ms-mus timescales. Based on these observations, we propose that the patch of conserved residues on the central face of the molecule corresponds to the site at which km23 binds the dynein intermediate chain and that the flexible cleft formed between the overlap of loops from the two monomers corresponds to the site at which km23 binds other partners, such as the TGFbeta type II receptor or Smad2.

Lanthanide(III) Complexes of a Mono(methylphosphonate) Analogue of H4dota: The Influence of Protonation of the Phosphonate Moiety on the TSAP/SAP Isomer Ratio and the Water Exchange Rate

A monophosphonate analogue of H4dota, 1,4,7,10-tetraazacyclododecane-4,7,10-tris(carboxymethyl)-1-methylphosphonic acid (H5do3aP), and its complexes with lanthanides were synthesized. Multinuclear NMR studies reveal that, in aqueous solution, lanthanide(III) complexes of the ligand exhibit structures analogous to those of H4dota complexes. Thus, the central ion is nine-coordinate, surrounded by four nitrogen atoms, three acetate and one phosphonate oxygen atoms, and one water molecule in an apical position. For complexes of H5do3aP with Ln(III) ions in the middle of the series, the abundance of the desired twisted square-antiprismatic (TSAP) isomer is higher than for the corresponding H4dota complexes. The TSAP/square-antiprismatic (SAP) isomer ratio is highly sensitive to protonation of the phosphonate group: a higher abundance of the TSAP isomer was found in acidic solutions. The microscopic protonation constants of the TSAP isomers are higher than those of the SAP isomers. The presence of one water molecule in the first coordination sphere of the complexes in the pH region studied (pH 2.5-7.0) is confirmed by 17O NMR spectroscopy. The results of a simultaneous fit of variable-temperature 17O NMR relaxation data and 1H NMRD profiles show that the residence time of water (tauM) in the Gd(III) complex is much smaller than for [Gd(dota)(H2O)]-. The exchange rate appears to be dependent on the pH of the solution. The values of tauM are 37, 40, and 14 ns at pH 2.5, 4.7, and 7.0, respectively. These observations can be explained by an extensive second-sphere hydrogen-bonding network that varies with the state of protonation of the phosphonate moiety. Upon protonation of the complex, the second-sphere hydration probably becomes more ordered, which may result in a decrease in penetrability and an increase in tauM. The relaxivity of the Gd(III) complex is almost independent of the pH and is equal to 4.7 s(-1) mM(-1) (20 MHz, pH 7 and 37 degrees C). The solid-state structure was determined for the Nd(III) complex. It crystallizes as the TSAP isomer and the unit cell contains two independent molecules of the complex with different Nd-O(water) bond lengths of 2.499 and 2.591 A.

Assigning uncertainties in the inversion of NMR relaxation data

Recovering the relaxation-time density function (or distribution) from NMR decay records requires inverting a Laplace transform based on noisy data, an ill-posed inverse problem. An important objective in the face of the consequent ambiguity in the solutions is to establish what reliable information is contained in the measurements. To this end we describe how upper and lower bounds on linear functionals of the density function, and ratios of linear functionals, can be calculated using optimization theory. Those bounded quantities cover most of those commonly used in the geophysical NMR, such as porosity, T 2 log-mean, and bound fluid volume fraction, and include averages over any finite interval of the density function itself. In the theory presented statistical considerations enter to account for the presence of significant noise in the signal, but not in a prior characterization of density models. Our characterization of the uncertainties is conservative and informative; it will have wide application in geophysical NMR and elsewhere.

Combination of inversion and fitting as an effective method for the analysis of NMR relaxation data

A method is proposed for analyzing the NMR relaxation data. The procedure includes using the nonnegative leastsquares (NNLS) to determine roughly the relaxation time values. Then those values are used as the initial parameters of the nonlinear fitting computation. As a result, accurate relaxation time data can be conveniently obtained without any preknowledge of the sample. This method is verified and demonstrated by computer simulations and NMR experimental results.

Determination of protein rotational correlation time from NMR relaxation data at various solvent viscosities

An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR. In the present work, an innovative approach to the determination of the protein rotational correlation time tau(R) from the heteronuclear relaxation data is proposed. The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution. The method has been tested on computer simulated relaxation data as compared to the traditional tau(R) determination method from T(1)/T(2) ratio. The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component. The resulting rotational correlation time of 5.56 +/- 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T(1)/T(2) ratio.

Solution Structure of the Ubiquitin-conjugating Enzyme UbcH5B

The ubiquitination pathway is the main pathway for protein degradation in eukaryotic cells. The attachment of ubiquitin to a substrate protein is catalyzed by three types of enzymes, namely a ubiquitin activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Here, the structure of the human ubiquitin-conjugating enzyme (E2) UbcH5B has been solved by a combination of homology modeling, NMR relaxation data and automated NOE assignments. Comparison to E2 structures solved previously by X-ray crystallography or NMR shows in all cases the same compact fold, but differences are observed in the orientation of both N and C-terminal alpha-helices. The N-terminal helix that is involved in binding to ubiquitin ligases (E3) displays a different position, which could have consequences for precise E2-E3 recognition. In addition, multiple conformations of the side-chain of Asn77 are found in solution, which contrasts the single hydrogen-bonded conformation in the crystal structures of E2 enzymes. The possible implication of this conformational freedom of Asn77 for its catalytic function is discussed.

Complexation and Self-Assembling of Sulfonatomethylated Calix[4]resorcinarene with Both Organic and Lanthanide Ions in Aqueous Media

The stoichiometry and binding constant of the paramagnetic lanthanide ion(Gd3+) with sulfonatomethylated calix[4]resorcinarene (H8Xna4) were evaluated from the NMR relaxation data. Both 1H NMR spectroscopy and NMR relaxation data indicate that interaction of tetramethylammonium (TEMA) and N-methylpyridinium (MePy) cations with H8Xna4 in the presence of Ln3+ (Lu3+ or Gd3+) results in theformation of ternary complexes [Ln(G)H8X] with lanthanide ions,coordinated via sulfonate groups and organic cation included intothe cavity of H8Xna4. The inclusion of long-chainedN-decyl-(DePy) and N-cetylpyridinium (CPy) ions into H8Xna4 cavity leads to self-assembling which can be revealed by NMR relaxation method with Gd3+ probe ions. The excess of alkylpyridinium or TEMA cations leads to disassembling of (Gd)n(H8X)m(RPy)maggregates.

Structural Studies on the Ca2+-binding Domain of Human Nucleobindin (Calnuc)

Nucleobindin, also known as calnuc, participates in Ca2+ storage in the Golgi, as well as in other biological processes that involve DNA-binding and protein-protein interactions. We have determined the three-dimensional solution structure of the Ca(2+)-binding domain of nucleobindin by NMR showing that it consists of two EF-hand motifs. The NMR structure indicates that the phi and psi angles of residues in both motifs are very similar, despite the noncanonical sequence of the C-terminal EF-hand, which contains an arginine residue instead of the typical glycine at the sixth position of the 12-residue loop. The relative orientation of the alpha-helices in the N-terminal EF-hand falls within the common arrangement found in most EF-hand structures. In contrast, the noncanonical EF-hand deviates from the average orientation. The two helix-loop-helix moieties are in the open conformation characteristic of the Ca(2+)-bound state. We find that both motifs bind Ca2+ with apparent dissociation constants of 47 and 40 microM for the noncanonical and the canonical EF-hand, respectively. The Ca(2+)-binding domain of nucleobindin is unstructured in the absence of Ca2+ and folds upon Ca2+ addition. NMR relaxation data and structural studies of the folded domain indicate that it undergoes slow dynamics, suggesting that it is floppier and less compact than a globular domain.

Treatment of Peak Intensity Uncertainties in NMR Relaxation Data Analysis Can Lead to Severe Artifacts

Accurate estimations of experimental uncertainties of relaxation rates are of vital importance for the interpretation of relaxation data, in particular for the Lipari-Szabo “model free” approach and for comparative relaxation studies. Here we report a systematic investigation of different methods for the estimation of experimental uncertainties on longitudinal R1 and transversal R2 rates using two different schemes for sampling the rates. We show that certain combinations of sampling strategies and methods of estimating experimental uncertainties result in wrong rates and rate errors. Practical recommendations for obtaining proper rate and rate uncertainties are deduced.

Dynamics of an Amorphous Polymer by an Improved NMR Approach Based on the Simultaneous Analysis of 1H and 13C Relaxation Times

A new approach to extract quantitative dynamic information from NMR relaxation data of amorphous polymers is presented, consisting of the simultaneous fitting of 1H and 13C T1 vs temperature curves obtained at different frequencies by means of unified motional models. The reliability of the dynamic parameters obtained by this approach is substantially increased with respect to the single curve analysis because of the possibility of both investigating motions over a wide frequency range and combining relaxation times carrying either global (1H nuclei) or local (13C nuclei) dynamic information. Experimental data were obtained for an amorphous ethylene−propylene random copolymer over a wide temperature range above its glass-transition: 1H spin−lattice relaxation times were measured by wide-line techniques at four Larmor frequencies, while high-resolution MAS techniques were used to get 13C spin−lattice relaxation times at a single frequency. The experimental data were analyzed in terms of segmental main-cha...

Aggregation and counter ion binding ability of sulfonatomethylcalix[4]resorcinarenes in aqueous solutions

1 H NMR spectroscopy, 1 H NMR spin-lattice relaxation, conductivity and pH-metric titration data have been used to study the aggregation and counter-ion binding of tetrasulfonatomethylcalix[4]resorcinarenes with methyl (H 8XNa 4) and amyl (H 8YNa 4) substituents. The data obtained reveal the self-aggregation of H 8YNa 4 driven by hydrophobic interactions. The NMR relaxation data indicate that Gd 3+ is bound by both H 8XNa 4 and H 8YNa 4. The Gd 3+ ions were found to induce the aggregation of H 8YNa 4 with critical association concentration (CAC) value less than in case of its self-aggregation. Using Gd 3+ as a probe of counter-ion binding with the aggregate interface it was found out that binding of quaternary ammonium cations (QUATSs) with H 8YNa 4 aggregates and SDS aggregates is quite different, while binding of inorganic ions by both types of aggregates is similar. The most outstanding peculiarity of QUATSs binding with H 8YNa 4 aggregates is selective recognition of QUATSs, possessing trimethylammonium group, detected by NMR relaxation method.

Specific Features of the Adsorption and Nuclear Magnetic Relaxation of the Water Molecules in Active Carbons: 2. The State of Water in Active Carbon with Relatively Large Pores According to the NMR Relaxation Data

The state of water upon adsorption on FAS-3 active carbon with relatively large micropores is studied by the NMR relaxation method. The dependences of the times of spin–lattice (T1) and spin–spin (T2) NMR relaxation of adsorbed water molecules on the adsorption value are established. The character of the dependences of T1 and T2 on the number of adsorbed water molecules per primary adsorption site reflects the specific features of the volume filling of micropores and the formation of a continuous adsorption layer on the mesopore surface due to cluster coalescence on the one wall of a pore. The results obtained are compared with the data for typical microporous active carbons, as well as with the data obtained by the adsorption method.

Efficient and accurate determination of the overall rotational diffusion tensor of a molecule from 15N relaxation data using computer program ROTDIF

We present a computer program ROTDIF for efficient determination of a complete rotational diffusion tensor of a molecule from NMR relaxation data. The derivation of the rotational diffusion tensor in the case of a fully anisotropic model is based on a six-dimensional search, which could be very time consuming, particularly if a grid search in the Euler angle space is involved. Here, we use an efficient Levenberg–Marquardt algorithm combined with Monte Carlo generation of initial guesses. The result is a dramatic, up to 50-fold improvement in the computational efficiency over the previous approaches [Biochemistry 38 (1999) 10225; J. Magn. Reson. 149 (2001) 214]. This method is demonstrated on a computer-generated and real protein systems. We also address the issue of sensitivity of the diffusion tensor determination from 15N relaxation measurements to experimental errors in the relaxation rates and discuss possible artifacts from applying higher-symmetry tensor model and how to recognize them.

Time-Dependent Diffusion and Surface-Enhanced Relaxation in Stochastic Replicas of Porous Rock

Understanding the connection between pore structure and NMR behavior of fluid-saturated porous rock is essential in interpreting the results of NMR measurements in the field or laboratory and in establishing correlations between NMR parameters and petrophysical properties. In this paper we use random-walk simulation to study NMR relaxation and time-dependent diffusion in 3D stochastic replicas of real porous media. The microstructures are generated using low-order statistical information (porosity, void–void autocorrelation function) obtained from 2D images of thepore space. Pore size distributions obtained directly by a 3D pore space partitioning method and indirectly by inversion of NMR relaxation data are compared for the first time. For surface relaxation conditions typical of reservoir rock, diffusional coupling between pores of different size is observed to cause considerable deviations between the two distributions. Nevertheless, the pore space correlation length and the size of surface asperity are mirrored in the NMR relaxation data for the media studied. This observation is used to explain the performance of NMR-based permeability correlations. Additionally, the early time behavior of the time-dependent diffusion coefficient is shown to reflect the average pore surface-to-volume ratio. For sufficiently high values of the self-diffusion coefficient, the tortuosity of the pore space is also recovered from the long-time behavior of the time-dependent diffusion coefficient, even in the presence of surface relaxation. Finally, the simulations expose key limitations of the stochastic reconstruction method, and allow suggestions for future development to be made.

Nuclear magnetic relaxation study of the microstructure of a bicontinuous cubic phase

A NMR relaxation and NMR translational diffusion study is performed on a bicontinuous cubic phase of the dodecyltrimethylammonium chloride/2H2O system with low water content. It is demonstrated how NMR relaxation measurements are combined with structural investigations of X-ray and NMR molecular diffusion measurements to create a consistent picture of the mesoscopic structure of the cubic phase. The 14N, spin–lattice (R1) and spin–spin relaxation rates (R2), were measured for the surfactant at two different field strengths and at four temperatures. The water and the surfactant translational diffusion coefficients were measured by NMR field gradient technique. The experimental spin relaxation rates, and translation diffusion coefficients were interpreted in a model based on a Brownian dynamics (BD) simulation of translational diffusion along curved interfaces (P. Hakansson, L. Persson and P.-O. Westlund, J. Chem. Phys., 2002, 117(19), 8634–8643, ). The model surfaces used in our BD relaxation model are also frequently used in interpretations of X-ray crystallography and in free energy calculations on bicontinuous phases. One of our BD-models was able to quantify the influence of the water/lipid interface on the NMR relaxation and diffusion data. Together with X-ray data, a consistent picture of the microstructure for this system was obtained.

Orientations of the principal components of electric field gradients and internal motions in dihydrogen ligands from the 2H T1 NMR relaxation data in solution.

The deuterium spin-lattice relaxation times in (D2) ligands of W, Ru and Os complexes are reviewed and analyzed in terms of the fast internal (D2) motions: free rotation, librations and 180 degrees jumps. The analysis was performed using quadrupolar coupling constant (DQCC) parameters taken from the solid-state 2H NMR spectra and density function theory calculations. It is shown that the calculated DQCC values can be corrected for further use in interpretations of deuterium relaxation times for Ru and Os dihydrogen complexes. The resulting data led to a criterion for using the relaxation data to distinguish fast-spinning dihydrogen ligands. It is shown that the principal components of electric field gradient tensors at D in the dihydrogen ligands are oriented closer to M-D directions.

Crystal Structures of Engrailed Homeodomain Mutants: IMPLICATIONS FOR STABILITY AND DYNAMICS

We report the crystal structures and biophysical characterization of two stabilized mutants of the Drosophila Engrailed homeodomain that have been engineered to minimize electrostatic repulsion. Four independent copies of each mutant occupy the crystal lattice, and comparison of these structures illustrates variation that can be partly ascribed to networks of correlated conformational adjustments. Central to one network is leucine 26 (Leu26), which occupies alternatively two side chain rotameric conformations (-gauche and trans) and different positions within the hydrophobic core. Similar sets of conformational substates are observed in other Engrailed structures and in another homeodomain. The pattern of structural adjustments can account for NMR relaxation data and sequence co-variation networks in the wider homeodomain family. It may also explain the dysfunction associated with a P26L mutation in the human ARX homeodomain protein. Finally, we observe a novel dipolar interaction between a conserved tryptophan and a water molecule positioned along the normal to the indole ring. This interaction may explain the distinctive fluorescent properties of the homeodomain family.

Phase Composition of Block Copoly(ether ester) Thermoplastic Elastomers Studied by Solid-State NMR Techniques

The microphase composition of poly(butylene terephthalate)-block-poly(tetramethylene oxide) multiblock (PBT-block-PTMO) copolymers with varying content of PTMO soft block and varying block length was studied by 1H NMR transverse magnetization relaxation (T2 relaxation) and 1H 2D DQ NMR experiments under fast MAS. The 2D NMR method showed the existence of the molecular scale mixing of PBT and PTMO blocks. The 1H and previously obtained 13C NMR relaxation data provided detailed information on the microphase composition. The content of soft domains (i.e., viscoelastic material which is composed of the soft block and mixed PBT) and hard domains (i.e., crystalline PBT and immobilized PBT chain portions at the crystal−amorphous PBT interface) depends largely on temperature. It was shown that about 15−25% of PBT in the samples is soft due to molecular mixing with PTMO. The results suggest that at 40 °C the mixed phase is composed of approximately one PBT chain unit per eight PTMO chain units. The PBT crystallinity in the copolymers slightly increases with increasing length of the soft block, since this causes increase of the average length of PBT blocks. The content of crystalline PBT is nearly constant for samples with hard blocks containing more than five monomer units and decreases for shorter PBT block lengths. An increase in the content of PTMO with the same block length causes a significant increase in chain mobility in the soft domains. Apparently, the content of the mixed phase and the density of physical/topological junctions at the PBT/PTMO interphase largely affect the molecular mobility in the soft domains.

Temperature Dependence of Domain Motions of Calmodulin Probed by NMR Relaxation at Multiple Fields

Interdomain motions of Ca(2+)-ligated calmodulin were characterized by analyzing the nuclear magnetic resonance (15)N longitudinal relaxation rate R(1), transverse relaxation rate R(2), and steady-state {(1)H}-(15)N NOE of the backbone amide group at three different magnetic field strengths (18.8, 14.1, and 8.5 T) and four different temperatures (21, 27, 35, and 43 degrees C). Between 35 and 43 degrees C, a larger than expected change in the amplitude and the time scale of the interdomain motion for both N- and C-domains was observed. We attribute this to the shift in population of four residues (74-77) in the central linker from predominantly helical to random coil in this temperature range. This is consistent with the conformation of these residues in the calmodulin-peptide complex, where they are nonhelical. The doubling of the disordered region of the central helix (residues 78-81 at room temperature) when temperature is raised from 35 to 43 degrees C results in larger amplitude interdomain motion. Our analysis of the NMR relaxation data quantifies subtle changes in the interdomain dynamics and provides an additional tool to monitor conformational changes in multidomain proteins.

NMR properties of alginate microbeads

Alginates are a family of unbranched polysaccharides with properties that vary widely depending on their composition. In the presence of multivalent cations (frequently Ca 2+), alginates form a gel. Consequently, alginates have been used to encapsulate a variety of biological materials, including cells. In this study, we present NMR relaxation and diffusion data from alginate microbeads with similar size and properties to those used in the development of a bioartificial pancreas. Our data demonstrate that the transverse relaxation time ( T 2) of water within the gel depends on the guluronic acid content of the alginate, whereas the longitudinal relaxation time ( T 1) and the apparent diffusion coefficient of water do not. Our data further suggest that the diffusion of Ca 2+ ions is hindered by the presence of a poly- l-lysine layer, a layer commonly added to provide mechanical support to the beads and immunoprotection to the encapsulated cells in the event of implantation. The impact of these data on our understanding of the role of alginate gels in the development of a bioartificial pancreas is discussed.